Ewing's sarcoma is a common pediatric tumor that arises in bone and soft tissues. Chromosomal translocations create fusion proteins between the N-terminus of EWS and the DNA binding domain from the closely related ets proteins Fli or Erg in at least 95% of Ewing's sarcomas. Related fusion proteins have been identified in five other types of human sarcomas and in myeloid leukemia. The EWS/Fli and EWS/Erg chimeric proteins can function as transcription factors and they can transform NIH-3T3 cells. Transcription activation and cellular transformation appear to be inter- related, but distinct functions of the fusion proteins. The exact process by which EWS/ets fusion genes contribute to tumorigenesis is unknown. The proposed studies will investigate the hypothesis that the EWS domain contributes unique properties to the chimeric proteins that play a critical role in the genesis of Ewing's sarcomas through interactions with other cellular proteins. In preliminary studies leading to this application, five proteins that interact with the tumor- associated domain of EWS were isolated multiple time in a yeast two- hybrid assay. Several lines of in vitro and in vivo experimentation are proposed to identify functional components of the EWS domain and determine the effects of protein-protein interactions on EWS/Fli activity. EWS-interacting proteins will be evaluated for their effect on transactivation and cellular transformation by EWS/Fli using cotransfection and cell culture assays. In addition, deletions and point mutations in the domain of EWS will be tested to define the residues required for protein-protein associations as detected by in vitro and cell culture assays. EWS/Fli proteins containing mutations in the interaction domains will be analyzed to determine the contribution of protein-protein interactions to transactivation and cellular transformation by EWS/Fli. Finally, we will identify the cell-types that are susceptible to transformation by EWS/Fli and study tumorigenesis in vivo using transgenic mice. Expression of functionally relevant EWS- interacting genes will be examined in Ewing's sarcoma cell lines and primary Ewing's sarcoma tissue. The results of these studies should provide a better understanding of the role of EWS/ets chimeric proteins in Ewing's sarcoma, and may identify common factors that contribute to tumorigenesis in human sarcomas and leukemias containing related fusionproteins.